This Project will contribute to the overall goals of the Program by investigating the arm of TCR signaling that activates NF-KB, a transcription factor that is required for antigen-induced T cell proliferation and activation. The study of TCR signaling to NF-KB offers several opportunities to expand our understanding of how a T cell interprets antigenic inputs. First, the mechanistic understanding of how the TCR activates NF-KB is far from complete. Critical components of this pathway remain undiscovered and it is likely that new players in this pathway will also play important roles in other arms of TCR signaling studied in the Program. In AIM 1, we will use a novel expression cloning strategy to identify enhancers and suppressors of TCR signaling to NF-KB. Second, maximal TCR-mediated NF-KB activation requires both TCR engagement by MHC plus antigen (signal 1) and costimulatory signals (signal 2). In AIM 2, we will test the hypothesis that the CARD11- GADS interaction is required for CD28-mediated costimulatory signaling to NF-KB. Third, while it is clear that molecules that signal from the TCR to NF-KB are recruited to the immunological synapse (IS) in a dynamic, regulated manner, it is unclear how and why this is precisely accomplished. In AIM 3, we will investigate how NF-KB signaling intermediates are recruited to the immunological synapse. This project will benefit from synergy with other projects in the Program. AIM 1 will use Core C and should yield novel components or modulators of TCR signaling pathways that can be studied in Projects 1, 2, 3, and 4 for roles in TCR clustering, Immunological Synapse (IS) formation and regulation, Sprouty!-mediated regulation, and calcium signaling, respectively. AIM 2 may offer molecular insight into how a T cell makes the cellular choice of activation or anergy, and will apply a mouse model and the expertise of J. Powell (Project 3). AIM 3 will use Core B and technology developed by A. Kupfer (Project 2) and will contribute to the understanding of IS formation and structure during T cell activation. Our results should add to the understanding of how the molecular machinery of immune cells can recognize and interpret environmental cues, including pathogenic and nonpathogenic stimuli, and respond appropriately. Since the inappropriate response to stimuli can result in ineffective immune surveillance, autoimmunity, or cancer, our results may yield molecular targets for new therapies designed to treat diseases of the immune system.